PROJECT SUMMARY The human gastrointestinal tract is colonized by a large and diverse population of bacteria and other microorganisms that are positive health assets needed for normal neural and immune development, nutrition, and metabolism. Conversely, it is now well-known that members of the gut microbiota are relevant to the etiology of numerous complex human diseases including impaired cognitive development, neurological diseases, obesity, diabetes, and cardiovascular disease. Current evidence suggests that the link between the gut microbiota and the host is in part mediated by vagal afferents that richly innervate the intestinal mucosa. Vagal afferent neurons express receptors for many known bacterial metabolites and thus represent a major target for the dissemination of information from the gut microbiota to the brain and other organ systems. However, understanding of the precise interactions between the microbiota, the intestinal epithelium and vagal afferent neurons is lacking. The current barrier to progress is the complexity of this interface and difficulty of confounding factors present in in vivo models. Currently, there are no models to adequately model the gut metabolome-epithelial barrier-vagal afferent neuron axis; thereby, identification of bacterial metabolites (particularly in response to dietary supplements) that are causative for either the promotion or detriment of human health is critically lacking. The overarching goal of this proposal is to bridge microfabrication technology, microbiology, and gastrointestinal-/neurophysiology to establish a novel in vitro platform for identifying bacterial metabolites that signal to gut sensory neurons (vagal afferents) via interaction with the intestinal epithelium. Specifically, the model is designed to: (i) sustain dissociated vagal afferent neurons in axonal contact with gut epithelial cells; (ii) isolate the two cultures so that soluble factors can be introduced to and contained in a single chamber; (iii) have microchannels interconnecting the two cultures via axonal structures; and (iv) have multiple electrode arrays in both chambers for electrophysiological monitoring and stimulation of neural cultures, as well as assessment of gut epithelial barrier function via trans-epithelial electrical resistance measurements. In order to achieve the project goals, the interdisciplinary research team will (i) establish a microfluidic culture platform for vagal afferent neurons; (ii) assess the influence of bacterial metabolites with a microfluidic epithelial cell culture; and (iii) engineer the epithelial cell-afferent neuron unit to study the influence of purified bacterial metabolites and sterile-filtered gut microbiota secretome from mouse-fed with different dietary regimens (e.g., low-/high-fat, high-sugar, dietary supplements, probiotics). Taken together, this platform will pioneer a new paradigm to study gut microbiota and the influence of a complementary dietary health approaches, and subsequently enable high-throughput screening of metabolites and therapeutics relevant to the gut-brain axis.